华北农学报 ›› 2022, Vol. 37 ›› Issue (4): 206-211. doi: 10.7668/hbnxb.20192971

所属专题: 薯类作物 植物保护 生物技术

• 资源环境·植物保护 • 上一篇    下一篇

甘薯4种RNA病毒多重RT-PCR检测方法的建立

冯丽婷, 张剑峰 , 迟胜起   

  1. 青岛农业大学 植物医学学院,山东 青岛 266109
  • 收稿日期:2022-02-22 出版日期:2022-08-28
  • 通讯作者: 张剑峰, 迟胜起
  • 作者简介:

    冯丽婷(1995-),女,宁夏中卫人,在读硕士,主要从事甘薯病毒研究。

  • 基金资助:
    山东省现代农业产业技术体系薯类产业体系(SDAIT-16-06)

Establishment of a Multiplex RT-PCR Assay for Detection of Four Sweet Potato RNA Viruses

FENG Liting, ZHANG Jianfeng , CHI Shengqi   

  1. College of Plant Health & Medicine,Qingdao Agricultural University,Qingdao 266109,China
  • Received:2022-02-22 Published:2022-08-28
  • Contact: ZHANG Jianfeng, CHI Shengqi

摘要:

针对甘薯生产上发生为害严重的甘薯RNA病毒-甘薯羽状斑驳病毒(SPFMV)、甘薯褪绿矮化病毒(SPCSV)、甘薯G病毒(SPVG)和甘薯C病毒(SPVC),建立了多重RT-PCR检测方法。首先根据GenBank中收录的这些病毒的外壳蛋白(CP)基因的保守序列,利用Premier 5.0 软件分别设计并合成了上述4种病毒的特异性引物;以4种病毒的下游特异性引物合成的cDNA为模板,在同一体系中同时加入上述4种甘薯病毒引物进行PCR扩增,初步建立这4种甘薯RNA病毒的最佳多重RT-PCR体系;通过对该多重RT-PCR体系的退火温度、延伸时间、dNTPs量、模板量、引物浓度等条件的优化,建立了能够同时检测这4种甘薯RNA病毒的最优四重RT-PCR检测体系,并对优化的多重RT-PCR体系进行了挑战测试和灵敏度测试。试验结果显示,优化的多重RT-PCR体系,各病毒引物之间不会出现交叉干扰,所扩增的4条目的条带清晰,能够明确区分上述4种甘薯病毒;挑战检测和灵敏度测试显示,此检测体系特异性强且灵敏度高。所建立的上述4种甘薯RNA病毒的多重RT-PCR检测方法能同时检测4种甘薯病毒,不仅准确灵敏,而且降低了检测成本,提高了病毒检测效率。

关键词: 甘薯, SPFMV, SPCSV, SPVG, SPVC, 多重RT-PCR

Abstract:

A multiplex RT-PCR detection system was established and studied to target four sweet potato RNA viruses,Sweet potato feathery mottle virus (SPFMV),Sweet potato chlorotic stunt virus (SPCSV), Sweet potato virus G (SPVG)and Sweet potato virus C (SPVC),which caused serious damage in sweet potato production. Firstly,the specific primers for four viruses were designed by Premier 5.0 software and synthesized according to the conserved sequences of coat protein(CP)gene of these viruses from GenBank. The cDNA synthesized was used as a template,and 4 couples of selected primers were used simultaneously to amplify the target fragments by PCR. A best multiplex RT-PCR reaction system was tested by optimizing the annealing temperature,extension time,dNTPs amount,template amount,primer concentration and so on and an optimal quadruple RT-PCR system that could simultaneously detect these four sweet potato RNA viruses was established. The results showed that an accurate and sensitive multiplex RT-PCR method for detecting the four sweet potato viruses mentioned above with respective target band had been successfully established by challenge tests in which there would be no crossover among the virus primers. The multiplex RT-PCR method established in this study could detect four kinds of sweet potato virus at the same time. It was not only accurate and sensitive,but also reduced the cost of detection and improved the efficiency of virus detection.

Key words: Sweet potato, Sweet potato feather mottle virus (SPFMV), Sweet potato chlorosis stunt virus (SPCSV), Sweet potato virus G (SPVG), Sweet potato virus C (SPVC), Multiplex RT-PCR

引用本文

冯丽婷, 张剑峰, 迟胜起. 甘薯4种RNA病毒多重RT-PCR检测方法的建立[J]. 华北农学报, 2022, 37(4): 206-211. doi: 10.7668/hbnxb.20192971.

FENG Liting, ZHANG Jianfeng, CHI Shengqi. Establishment of a Multiplex RT-PCR Assay for Detection of Four Sweet Potato RNA Viruses[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(4): 206-211. doi: 10.7668/hbnxb.20192971.

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